In the presence of ATP, the Escherichia coli RecA protein, or a homologous recombination protein from another organism, catalyzes strand exchange between a number of substrates, including between single- and double-stranded DNAs. A RecA protein coated single-stranded oligonucleotide interacts with double-stranded target nucleic acid sequence homologous to the oligonucleotide in a process called “synapsis.” During synapsis an intermediate containing hybridized, partially joined molecules is formed followed by branch migration to form fully hybrid molecules between the original single- and double-stranded DNAs. The extent to which the nucleic acid molecules hybridize is dependent upon the extent of their homology and hybridization between two nucleic acids that are not completely complementary is stabilized in the presence of RecA.
RecA protein can form stable complexes with short oligonucleotides, at least approximately 9 nucleotides in length, in the presence of the non-hydrolyzable ATP analog, ATP-γ-S. These RecA coated nucleic acid complexes can then be mixed with target double-stranded nucleic acid to form triplex nucleic acid structures or “single D-loops,” which are extremely unstable in the absence of RecA. Upon removal of RecA protein the single D-loop joints are stable in supercoiled but very unstable in relaxed DNA.
Addition of a second oligonucleotide that recognizes the strand of the target nucleic acid opposite to the first oligonucleotide results in the formation of a complement-stabilized or “double” D-loop structure. Although this structure is more stable after removal of RecA than the single D-loop structure, the stability depends on the length of the oligonucleotides, and when two DNA oligonucleotides are used the oligonucleotides generally must be at least about 80 nucleotides. See, for example, U.S. Pat. No. 5,670,316, which is incorporated herein by reference in its entirety. Previous efforts to enhance the stability of a double D-loop after removal of RecA involve complex dideoxyoligonucleotides which comprise unusual secondary structures. See, for example, international patent application WO 00/63365, which is incorporated herein by reference in its entirety.
When a linear, double-stranded nucleic acid target and oligonucleotides homologous to the end of the target are used, the structures that are formed at the end of the linear duplex are a subcategory of double D-loops designated “Y-loops”. Y-loops can be formed by the same methods used to form double D-loops.
A need exists for stable oligonucleotide:target nucleic acid complexes made using simple, inexpensive oligonucleotides.